The present invention relates to inocula. More particularly, the present invention relates to inocula containing microorganisms of the genus Rhizobium and which have a low water activity, a long storage life and improved resistance to temperature and rehydration. The invention also relates to a method of preparing such inocula.
Numerous attempts have been made to preserve Rhizobium by dehydration employing various adjuvants, such as on dehydrated balls of porcelain (HUNT, 1958, J. Bacteriol. 76, 453-454); in suspension in dehydrated liquid oils (U.S. Pat. No. 3,034,968); as a dehydrated suspension in oils mixed with talc or kaolin (U.S. Pat. No. 3,168,796); by adsorption on dry granules of plaster (French Pat. No. 1,490,046); as a dehydrated powder on granules of gypsum (FRASER, 1975, J. Appl. Bacteriol. 39, 345); and by using sodium sulfate without water to dehydrate the Rhizobium by formation of a water of crystallization. (NILSSON, 1957, (NILSSON, 1957, cited in Method for Evaluating Biological Nitrogen Fixation, F. Bergersen, ed., 493). However, the dehydration processes disclosed in these patents and publications are slow and thus harmful to the survival of the microorganism (e.g., Gault in Newsletter, Vol. 21 (1) April 1981, page 34, and Vincent in Rhizobium, Newsletter, 25, 1981). The steps leading to the dehydrated state can cause the destruction of the Rhizobium.
Methods such as freeze-drying (U.S. Pat. No. 3,168,796) or spray drying (OCHIN, 1980, Dissertation Lille) which use different protective or support substances, such as lactose, milk, cystein hydrochloride, maltodextrin, or finely divided, chemically inert substances of the kaolin type, and activated charcoal, have also been employed. But even with the addition of these protective substances, the drying process, the preservation, and the rehydration are very difficult.
The rehydration of the dehydrated microorganism is particularly difficult. It is known that the progressive absorption of water by a totally dehydrated microorganism is lethal (Amarger, Arch. Mikrobiol 81, 361-366 (1972)). When, for instance, a freeze-dried ampule containing microorganisms is opened, all the microorganisms are dead in less than one week in ambient air. It is, therefore, possible to preserve a portion of the microorganisms by extensive dehydration, but when it is necessary to employ the dehydrated microorganisms in the open field, the use of a solution to rehydrate the microorganisms can create numerous problems.
Attempts have been made to obtain a microorganism enclosed in a polymer which can be preserved indefinitely without special conditions and which is in solid form at the time of its use. This problem has been difficult to solve, and it is believed by those skilled in the art that in order to assure the survival of the microorganisms, a certain amount of humidity must be maintained in the inocula. The culture medium can be added to a support, such as peat, or directly to a polymer gel.
U.S. Pat. No. 4,155,737 discloses the inclusion of a microorganism in a polymer gel which is a polyacrylamide gel or a silica gel. An attempt is made to reduce the water loss, or at least to maintain a water availability (or a water activity) in the inoculum. In addition to the microorganism, the inoculum contains soluble mineral elements, a carbohydrate source such as mannitol, and a nitrogen source such as yeast extract.
In practice, the polymer must be biodegradable or at least non-polluting. For this reason, European Application No. 17,565 discloses the use, as a support, of a matrix having a base of at least one polymer from the group of polysaccharides. This process is characterized by the fact that the gel which contains the microorganism is subjected to a drying, which leaves at least one part of bulk water held in the gel. This drying can be improved by the addition of a substance which has a strong absorption for water, such as a synthetic or natural silica.
In order to assure that a good percentage of the microorganisms survive, the water activity should generally be maintained at a value which is preferably above 0.85. However, at the optimum moisture content required in the inocula referred to in these patents, for instance for the survival of Rhizobium, the germination of fungi spores and the development of molds and contaminants is a problem with nonsterile products. For most molds, the water activity range within which development is possible is generally between 0.80 and 0.95, but certain molds which are referred to as xerophytic or "osmotolerant" are capable of developing at lower water activities (a.sub.w =0.6-0.7).
In order to assure the microbiological stability of inocula having an optimal water content, it has been necessary to prepare and store the inocula in sterile form or to subject the inocula to cold storage. The stability of the refrigerated inocula results both from the lowering of the temperature and from the blocking of a portion of the water available to the contaminants. As a general rule, these inocula remain unstable with respect to contaminants and poorly resist temperatures above about 40.degree. C., which is a drawback in hot climates or, in general, upon preservation, due to a definite risk of contamination.
In order to control the different degradation processes (i.e., development of microorganisms, chemical reactions, enzymatic reactions, changes in texture) it is possible to act more or less selectively on each of these phenomena by controlling the customary parameters of physical chemistry, temperature, pH, redox potential, and by the use of specific inhibitors. However, these means have proven limited. This is due, in particular, to the fact that the prior art adheres to the principle that the water available must be sufficient to assure the survival of the microorganism.
It has now been discovered that, starting from the customary water activity values of 0.85 and more in inocula, if the water activity is lowered, there is an initial decrease in the development of molds and of the enzymatic reactions but that substantial plasmolysis of the cells and browning reactions occur. Similar effects are noted in going from a very low water activity to a water activity of the order of 0.85 and more. As a matter of fact, the change to intermediate water activities on the order of 0.4 to 0.8 in lethal for bacterial, whether this change is effected upon dehydration or upon rehydration. In the case of dehydration, this phenomenon has been overcome by an extremly rapid passage through this lethal region by means of freeze-drying (U.S. Pat. No. 3,168,796 to Scott) or by means of spray drying. However, this technique does not solve the problem of destruction of the microorganism under conditions of use in which rehydration is slow and progressive.